寡核苷酸與原態蛋白質於聚環氧乙烯溶液之毛細電泳質譜研究

Capillary Electrophoresis / Electrospray Mass Spectrometry Analysis of Oligonucleotides and Native Proteins Using Poly(ethylene oxide) Solution

陳思坊(Szu-Fang Chen)

指導教授 ： 何國榮

國立臺灣大學/理學院/化學研究所/碩士(2011年)

https://doi.org/10.6342/NTU.2011.00439

摘要

利用毛細電泳分析寡核苷酸(oligonucleotides)與原態蛋白質(native protein)時，會在緩衝溶液中加入聚合物(polymer)，使淌度相近的分析物藉由篩分機制(sieving mechanism)達到分離。然而，聚合物會造成分析物訊號於電灑法質譜偵測的抑制與污染質譜游離源。為了減少聚合物對於分析物訊號於電灑法質譜偵測的抑制與污染質譜游離源，本研究利用液體接合式-低流速鞘流介面(liquid-junction/low flow interface)銜接使用聚合物溶液之毛細電泳-電灑法質譜分析寡核苷酸與原態蛋白質。分析帶負電的寡核苷酸時，系統操作於反向電場之下，而帶負電荷之寡核苷酸可藉由本身淌度大於電滲流的情況之下，往質譜端移動而被偵測。但不帶電之聚合物將隨電滲流往進樣端持續後退而使分離有效長度下降而影響分離效率。藉由於液體接合槽中裝填聚合物溶液，使聚合物能夠回補至分離管，以維持分析物的分離效率。分析帶正電荷原態蛋白質時，系統操作於正向電場之下，聚合物將隨電滲流往質譜端移動，因此使用聚乙烯醇(polyvinyl alcohol, PVA)塗佈之毛細管作為銜接管，控制銜接管內的電滲流為零，讓聚合物停留在液體接合槽中，可有效避免質譜游離源污染與分析物訊號的抑制，而分析物可藉由本身淌度往質譜端移動而被成功偵測。

關鍵字

聚合物溶液 ； 寡核苷酸 ； 原態蛋白質 ； 毛細電泳質譜 ； 液體接合式-低 ； 流速鞘流介面

並列摘要

Analyzing oligonucleotides and native proteins by capillary electrophoresis usually adds polymer in the background electrolyte. By using the polymer solution, analytes with similar mobilities can be separated via molecular sieving mechanism. However, PEO will suppress the analyte signal and contaminate the ESI ion source. To alleviate these problems resulting from the introduction of polymers into ESI source, a liquid junction-low flow interface was utilized to hyphenate CE with polymer solution and ESI-MS in the analysis of oligonucleotides and native proteins. In the analysis of negative charged oligonucleotides, polymers would migrate toward the inlet reservoir with EOF under reversed polarity to avoid the introduction of polymers into ion source. In addition, filling the liquid junction with polymer solution could make polymers in the liquid junction reservoir flow into separation column. Thus, the separation efficiency could be maintained. Because of the mobility of analyte is greater than EOF, so it could migrate to MS inlet and be successfully detected. On the other hand, polymers would migrate to MS inlet with EOF under normal polarity in the analysis of intact proteins. As a consequence, a PVA coated capillary column was utilized as the connecting column to make polymer stay in the liquid junction. By this method, the introduction of polymers into ion source could be prevented. And the analyte could migrate to the detection end and be detected by its own mobility.

並列關鍵字

oligonucleotides ； polymer solution ； PEO ； sieving mechanism ； CE-MS ； native protein ； liquid-junction/low flow interface

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